The long-term goal of the proposed experiments is the repair of damaged or degenerated cortico-spinal circuitry via i) induction of neurogenesis of new cortico-spinal motor neurons (CSMN) from endogenous precursors ("stem cells"); ii) support of their survival, and iii) recruitment into functional synaptic circuitry with the lower motor neuron population. Recently, we have manipulated endogenous precursors in situ in the adult mouse to undergo neurogenesis (birth of new neurons) de novo in adult mouse neocortex, where it does not normally occur. In collaborative work, we induced behaviorally functional neuronal replacement in situ in songbirds by homologous avian endogenous precursors. These experiments demonstrated that there exists a sequence and combination of molecular signals by which the birth of new neurons can be induced, even in the adult neocortex where neurogenesis does not normally occur. Even more directly relevant to this proposal are our recent findings that 1) endogenous precursors can also be specifically induced to differentiate in situ into cortico-spinal motor neurons (CSMN) with projections to the spinal cord; and 2) that we can isolate, FACS purify (to >99.5% purity), and culture CSMN at distinct developmental stages for analysis of lineage-specific controls over survival and differentiation, particularly rate / extent of axon elongation. We hypothesize that we can substantially increase the number of newly recruited CSMN by enhancing the survival of the newborn neurons and/or their rate and extent of axonal outgrowth to supportive spinal cord targets. We also hypothesize that newborn neurons can differentiate precisely into new functional CSMN, receive afferent synapses, and become synaptically integrated. Our five Aims will test these and related hypotheses directly, in vitro and in vivo. The proposed research will: Aim 1) undertake a more complete analysis of lineage-specific differentiation of the induced adult-born CSMN, and a more detailed characterization of the time course of induced neurogenesis, neuronal differentiation, connectivity, and long-term survival of newborn CSMN); Aims 2,3) investigate in vitro a select set of candidate growth and neurotrophic factors enabling potentially stage-specific enhanced survival and/or enhanced outgrowth and circuit connectivity of CSMN, employing FACS purification of CSMN at distinct developmental stages, and bath vs. localized factor application; Aim 4) manipulate and increase the induced adult CSMN neurogenesis and spinal projections by intraventricular infusion of highly selected candidate growth factors from the in vitro experiments and Aim 5) investigate the precision of CSMN differentiation and potential afferent and efferent synapse formation using ICC and retroviral GFP-barley lectin expression. Together, this work aims toward the ultimate goal of repair of cortico-spinal motor neuron circuitry by manipulation of endogenous neural precursors in situ, without transplantation.